Assay for detecting closely-related serotypes of human papillomavirus (HPV)

ABSTRACT

A real time Taq-Man PCR assay for detecting multiple serotypes of human papillomavirus (HPV) wherein the number of serotypes detected exceeds the number of colorimetric channels for detection. A biological sample is combined with three oligonucleotide primer/probe sets such that the probes and primers anneal to a target sequence. Each primer/probe set is at least preferential for a specific serotype of an organism. The first and second primer/probe sets are degenerate with respect to each other. The third primer/probe set is not degenerate with respect to the first and second primer/probe sets and discriminates for a third serotype. The third primer/probe set has a signal moiety that emits signal at a wavelength that is the same or different from the wavelength emitted by the signal moiety of the degenerate primer/probe set probes. The target sequences, if present, are amplified and detected.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Provisional Patent Application No. 61/304,941 filed Feb. 16, 2010, thedisclosures of which are hereby incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jan. 31, 2011 is named SequenceListing for Detecting Related Serotypes of HPV, and is 7.55 kilobytes insize.

BACKGROUND OF THE INVENTION

More than 80 types of human papillomaviruses (HPVs) have beenidentified. The different types of HPV cause a wide variety ofbiological phenotypes, from benign proliferative warts to malignantcarcinomas (for review, see McMurray et al., Int. J. Exp, Pathol. 82(1):15-33 (2001)). HPV6 and HPV11 are the types most commonly associatedwith benign warts, whereas HPV16 and HPV18 are the high-risk types mostfrequently associated with malignant lesions. Determination of thespecific type of HPV in a clinical sample is, therefore, critical forpredicting risk of developing HPV-associated disease.

Several nucleic acid-based methods have been utilized to identify andquantify specific HPV types in clinical samples, such as detection ofviral nucleic acid by in situ hybridization, Southern blot analysis,hybrid capture or polymerase chain reaction (PCR). The Hybrid Capture®II (Qiagen, Inc., Valencia, Calif.) assay utilizes antibody capture andnon-radioactive signal detection, but detects only a single target of agiven set of HPV types (See, e.g., Clavel et al., British J. Cancer80(9): 1306-11 (1999)). Additionally, because the Hybrid Capture® IIassay uses a cocktail of RNA probes (probe cocktails are available forhigh risk or low-risk HPV types), it does not provide information as tothe specific HPV type detected in a sample, but rather provides only apositive or negative for the presence of high-risk or low-risk HPV.Similarly, many PCR-based methods often involve amplification of asingle specific HPV target sequence followed by blotting the resultingamplicon to a membrane and probing with a radioactively labeledoligonucleotide probe.

Other methods exploit the high homology between specific HPV genes ofdifferent types through the use of commercially available consensusprimers capable of PCR amplification of numerous HPV types present in asample. The presence of a specific HPV type is then identified using atype-specific oligonucleotide probe. See, e.g., Kleter et al., Journalof Clinical Microbiology 37(8): 2508-2517 (1999); Gravitt et al.,Journal of Clinical Microbiology 38(1): 357-361 (2000). Similarly,assays that utilize degenerate PCR primers take advantage of thehomology between HPV types, allowing detection of a greater number ofHPV types than methods utilizing specific primer sets. See, e.g. Harwoodet al., Journal of Clinical Microbiology 37(11): 3545-3555 (1999). Suchassays also require additional experimentation to identify specific HPVtypes.

The PCR methods described above can be associated with several problems.For example, differences in reaction efficiencies among HPV types canresult in disproportionate amplification of some types relative toothers. Additionally, the equilibrium for amplification will be driventowards those types that exist at higher copy numbers in a sample, whichwill consume the PCR reaction components, thus making amplification ofthe minor HPV types less likely.

Also described in the art is a 5′ exonuclease fluorogenic PCR-basedassay (Taq-Man PCR) which allows detection of PCR products in real-timeand eliminates the need for radioactivity. See, e.g., U.S. Pat. No.5,538,848; Holland et al, Proc. Natl. Acad. Sci. USA 88: 7276-7280(1991). This method utilizes a labeled probe, comprising a fluorescentreporter (fluorophore) and a quencher that hybridizes to the target DNAbetween the PCR primers. Excitation of the fluorophore results in therelease of a fluorescent signal by the fluorophore which is quenched bythe quencher. Amplicons can be detected by the 5′-3′ exonucleaseactivity of the TAQ DNA polymerase, which degrades double-stranded DNAencountered during extension of the PCR primer, thus releasing thefluorophore from the probe. Thereafter, the fluorescent signal is nolonger quenched and accumulation of the fluorescent signal, which isdirectly correlated with the amount of target DNA, can be detected inreal-time with an automated fluorometer.

Taq-Man PCR assays have been adapted for HPV type detection. Swan et al.(Journal of Clinical Microbiology 35(4): 886-891 (1997)) disclose afluorogenic probe assay that utilizes type-specific HPV primers thatamplify a portion of the L1 gene in conjunction with type-specificprobes. The Swan et al. assay measures fluorescent signal at the end ofa fixed number of PCR cycles (endpoint reading) and not in real-time.

Josefsson et al. (Journal of Clinical Microbiology 37(3): 490-96 (1999))report a Taq-Man assay that targets a highly conserved portion of the E1gene in conjunction with type-specific probes labeled with differentfluorescent dyes. A number of HPV types were amplified by utilizing amixture of specific and degenerate primers. Josefsson et al. utilized upto three type-specific probes per assay, which were designed to detect aportion of the E1 gene from different HPV types. Unlike the Swan et al.assay, Josefsson et al. measured the accumulation of fluorescence inreal-time.

Tucker et al. (Molecular Diagnosis 6(1): 39-47 (2001)) describe an assaythat targets a conserved region spanning the E6/E7 junction. Like theJosefsson assay, Tucker et al. employed real-time detection andtype-specific fluorescent probes. Tucker et al. also utilized multiplexPCR to simultaneously detect HPV target sequences and either the actinor globin cellular loci in the same reaction tube.

One of the particular challenges with HPV detection is the fact thatthere are many HPV types of clinical interest. Although multiplex assaysfor HPV detection are known, the multiplex assays are limited by thenumber of colorimetric channels for detection. These channels arevarious wavelengths of light (or ranges or bands of wavelengths). Eachchannel detects a signal emitted by a signal moiety that emits light ata specific channel wavelength. The number of HPV types that are detectedis therefore limited by the number of different, distinctly detectablesignal moieties in the assay.

Despite the development of the HPV assays described above, it would beadvantageous to develop a multiplex assay that is highly sensitive andreproducible, and that requires reduced man-hours compared to methodsdisclosed in the art. Given the many HPV types, it would be useful todetect more HPV types than there are detection channels of the assay.

SUMMARY OF THE INVENTION

The present invention is directed to a real-time PCR amplification thatdeploys Taq-Man chemistry to simultaneously detect more types of HPVthan there are channels for detection. In other words, in an assay withN channels, the number of HPV types that are detected is at least N+1.

The assay deploys a primer/probe set for detection of each type of HPVthat the assay is configured to detect. Among the various serotypessusceptible for detection by the assay include, for example, HPV types33, 39, 51, 56, 58, 59, and 68. The skilled person will appreciate thatthe probe/primer sets described herein might be combined with otherprimer/probe sets for other types of HPV (e.g. 16, 18, and 45). At leastone type is detected using a single primer probe set. In the context ofPCR amplification using a Taq-Man assay, the primer probe set is atleast a forward primer, a reverse primer and a probe. At least two typesare selected by a pair of primer probe sets with oligonucleotidesequences that are degenerate with respect to each other. “Degeneratesequences” as used herein, are two oligonucleotide primers or probesthat are complementary to the same locus of the same gene of differentclosely related types and which have minor sequence variations withrespect to each other to make them discriminatory between the two types.

In one embodiment of the present invention, the degenerate primer probessets are for detection of HPV types 39 and 68. The primer/probe settargets the same locus on the E6 gene of both types. The target lengthis 91 nucleotides and is illustrated in FIG. 1. The target is only asegment of the entire E6 gene. The target for the degenerateprimer/probe set for HPV types 39 and 68 is SEQ ID NO. 36. In apreferred embodiment the signal moieties for both probes is a cyaninedye label commercially available as CY5.

In this embodiment, the primer probe set that is not degeneratediscriminates for HPV Type 51. The primer/probe set for type 51 alsotargets the E6 gene, although in a different locus. In a preferredembodiment the signal moieties for this probe in this primer probe setis FAM, which is specifically identified below.

In a second embodiment of the present invention, the degenerateprimer/probes sets are for detection of HPV types 33 and 58. Theprimer/probe set targets the same locus on the E6 gene of both types.The target length is 138 nucleotides and is illustrated in FIG. 2. Thetarget is only a segment of the entire E6 gene. The target for thedegenerate primer/probe set for HPV types 33 and 58 is SEQ ID NO. 37. Ina preferred embodiment the signal moieties for both probes is FAM.

In this embodiment, the primer probe set that is not degeneratediscriminates for HPV Type 59. The primer/probe set for type 59 alsotargets the E6 gene, but not necessarily the same locus. In a preferredembodiment the signal moieties for this probe in this primer probe setis CY5, which is specifically identified below.

In a third embodiment of the present invention, the degenerateprimer/probes sets are for detection of HPV types 56 and 66. Theprimer/probe set targets the same locus on the E6 gene of both types.The target length is 79 nucleotides and is illustrated in FIG. 3. Thetarget is only a segment of the entire E6 gene. The target for the HPVtype 56 and 66 degenerate primer/probe set is SEQ ID NO. 38. In apreferred embodiment the signal moieties for both probes is CY5.

In this embodiment, the primer probe set that is not degeneratediscriminates for HPV Type 59. The primer/probe set for Type 59 targetsthe E6 gene. In a preferred embodiment the signal moieties for thisprobe in this primer probe set is CY5, which is specifically identifiedbelow. In this embodiment, the same signal moiety that is used for HPVType 59 is also used for HPV Types 56_(—)66. Since the same dyes are onthe same channel, there is no optical discrimination between HPV types59 and 56 66 in this embodiment.

This embodiment contemplates a multiplex assay that deploys yet anotherprimer/probe set (either degenerate or discriminatory) for yet anotherHPV serotype (e.g. the degenerate primer/probe set for HPV serotypes 33and 58). The signal moiety for this other HPV serotype is not CY5, andemits at a wavelength that is detectably different from the emissionwavelength of Cy5 (e.g. FAM).

In other embodiments, the signal moiety for the HPV Type 59 probe isFAM, which can be optically distinguished from the CY5 signal moiety forthe degenerate primer/probe set for HPV serotypes 56 and 66.

Alternate embodiments contemplate an assay or a probe set or a kit withany and all combinations of the primer/probe sets and the degenerateprimer/probe sets described herein. Specifically, any of theprimer/probe sets for HPV serotype HPV 51 or HPV 59 can be combined withany of the degenerated primer/probe sets for HPV serotypes HPV 39_(—)68;HPV 33_(—)58; and HPV 56_(—)66. Specifically contemplated is an assay orprobe set or a kit which combines the primer/probe set for HPV 51 withthe degenerate primer probe set of one or more of HPV 39_(—)68; HPV33_(—)58; and HPV 56_(—)66. An assay or probe set or a kit whichcombines the primer/probe set for HPV 59 with the degenerate primerprobe set of one or more of HPV 39_(—)68; HPV 33_(—)58; and HPV56_(—)66. Embodiments where the signal moieties for the probes are thesame or different dyes are contemplated. Since a multiplex assay iscontemplated, the primer/probe sets can be combined with otherprimer/probe sets configured to detect the presence or absence of otherserotypes of HPV. In those embodiments where the signal moiety for thedetector probe in the degenerate primer/probe sets is the same as thesignal moiety for the primer/probe set for HPV serotype 51 or 59, it iscontemplated that the multiplex assay would include a fourthprimer/probe set for a fourth serotype of HPV (e.g. HPV 31, HPV 52, HPV45, etc.) and that the fourth primer/probe set would have a signalmoiety that is different from the signal moieties for the detectorprobes of the degenerate primer/probe sets and the HPV 51 or 59primer/probe sets. Also, contemplated herein are multiplex assays inwhich two degenerate primer/probe sets are combined in one assay. Forexample, in one microwell, a first degenerate primer/probe set fordetecting the present of HPV serotypes 33 and 58 can be combined with asecond degenerate primer/probe set for detecting the presence or absenceof HPV serotypes 56 and 66. In these embodiments the probes of the firstdegenerate primer probe set has a signal moiety that emits at wavelengththat is detectably different from the emission wavelength of the signalmoiety for the second degenerate primer/probe set. In this example thesignal moiety for HPV serotypes 33 and 58 probes would be FAM and thesignal moiety for HPV serotypes 56 and 66 probes would be CY5.

In addition to the signaling moieties described above, the probes alsohave a non-fluorescent dark quencher. One example of a suitable darkquencher is BHQ™ 1 (Biosearch Technologies), which is suited for usewith the FAM fluorophore. Another example of a suitable quencher is BHQ™2 (Biosearch Technologies), which is suited for use with the CY5fluorophore. Other examples of signaling moieties are well known tothose skilled in the art and are not described in detail herein.

As used herein, the term “primer” refers to an oligonucleotide that iscapable of acting as a point of initiation of synthesis along acomplementary strand when placed under conditions in which synthesis ofa primer extension product which is complementary to a nucleic acidstrand is catalyzed. Such conditions include the presence of fourdifferent deoxyribonucleoside triphosphates and apolymerization-inducing agent such as DNA polymerase or reversetranscriptase, in a suitable buffer (“buffer” includes components whichare cofactors, or which affect ionic strength, pH, etc.), and at asuitable temperature. As employed herein, an oligonucleotide primer canbe naturally occurring, as in a purified restriction digest, or beproduced synthetically. The primer is preferably single-stranded formaximum efficiency in amplification.

As used herein, “primer pair” refers to two primers, a forward primerand a reverse primer, that are capable of participating in PCRamplification of a segment of nucleic acid in the presence of a nucleicacid polymerase to produce a PCR amplicon. The primers that comprise aprimer pair can be specific to the same HPV gene, resulting in anamplicon that consists of a sequence of nucleotides derived from asingle HPV gene. Alternatively, the primers that comprise a primer paircan be specific to different HPV genes that reside within closeproximity to each other within the HPV genome, thereby producingamplicons that consist of a sequence of nucleotides derived from morethan one gene.

As used herein, “different imaging spectra,” in reference to thefluorophores of the present invention, means that each fluorophore emitsenergy at a differing emission maxima relative to all other fluorophoresused in the particular assay. The use of fluorophores with uniqueemission maxima allows the simultaneous detection of the fluorescentenergy emitted by each of the plurality of fluorophores used in theparticular assay.

As used herein, the term “discriminatory,” used in reference to theoligonucleotide primers and probes of the present invention, means thatsaid primers and probes are specific to a single HPV type. It includesHPV primers and probes specific to a single HPV type, but that sharesome homology with other HPV types. “Discriminatory” primers and probesof the present invention include those oligonucleotides that lack 3′homology with other HPV types in at least one nucleotide or more. Such aresidue that is unique for the specific HPV type at the specificposition and acts to discriminate the HPV type from the others in thealignment is referred to as a “discriminatory base”. The term“discriminatory,” in reference to oligonucleotides, does not includeprimers and probes that are specific to more than one HPV type, i.e.those that share full homology with greater than one HPV type. In thisregard the degenerate primer/probe sets disclosed herein do notdiscriminate for the HPV type for which they are configured. Forexample, for the degenerate primer/probe set that targets HPV types 39and 68, the primer/probe set for HPV type 39 has a preference for HPVtype 39 over HPV type 68 but does not discriminate for HPV type 39 overHPV type 68. Because the degenerate primer/probe sets do notdiscriminate between the HPV types, the probes preferably have the samesignal moiety. Since the assay is configured to determine the presenceor absence of these HPV types in the aggregate, there is no need to usetwo optical channels for this purpose. Furthermore, when the degenerateprimer/probe set is combined with other primer/probe sets in a singlemicrowell for a multiplex assay, the signal moieties for the otherprimer/probe sets (either discriminatory or non-discriminatory) can bethe same or different. The signal moieties for the assay are largely amatter of design choice. The skilled person will use the same signalmoieties for primer/probe sets in the same microwell when it isnecessary to know if the particular HPV type is present. The same signalmoiety may be used on primer/probe sets for different HPV serotypes whenit is sufficient to know if only one of those types is present.

As used herein, “amplicon” refers to a specific product of a PCRreaction, which is produced by PCR amplification of a sample comprisingnucleic acid in the presence of a nucleic acid polymerase and a specificprimer pair. An amplicon can consist of a nucleotide sequence derivedfrom a single gene of a single HPV type or an amplicon can consist of anucleotide sequence derived from more than one gene of a single HPVtype.

As used herein, “primer/probe set” refers to a grouping of a pair ofoligonucleotide primers and an oligonucleotide probe that hybridize to aspecific nucleotide sequence of a single HPV type. Said oligonucleotideset consists of: (a) a forward discriminatory primer that hybridizes toa first location of a nucleic acid sequence of an HPV type; (b) areverse discriminatory primer that hybridizes to a second location ofthe nucleic acid sequence of the HPV type downstream of the firstlocation and (c) a fluorescent probe labeled with a fluorophore and aquencher, which hybridizes to a location of the nucleic acid sequence ofthe HPV type between the primers. In other words, an oligonucleotide setconsists of a set of specific PCR primers capable of initiatingsynthesis of an amplicon specific to a single HPV type, and afluorescent probe which hybridizes to the amplicon.

As used herein, “plurality” means two or more.

As used herein, “specifically hybridizes,” in reference tooligonucleotide sets, oligonucleotide primers, or oligonucleotideprobes, means that said oligonucleotide sets, primers or probeshybridize to a nucleic acid sequence of a single HPV type.

As used herein, “gene” means a segment of nucleic acid involved inproducing a polypeptide chain. It includes both translated sequences(coding region) and 5′ and 3′ untranslated sequences (non-codingregions) as well as intervening sequences (introns) between individualcoding segments (exons). For purposes of the description of theembodiments of the present invention, the HPV genome has a plurality ofgenes: e.g., L1, L2, and E1, E2, E4-E7.

As used herein, “locus” refers to the position on a chromosome at whichthe gene for a trait resides. The term locus includes any one of thealleles of a specific gene. It also includes homologous genes fromdifferent HPV types. For example, PCR assays that detect the L1 gene inHPV16 and HPV6 are single-locus assays, despite the detection ofsequences from different HPV types. Contrarily, for example, assays thatdetect the L1 gene and the E1 gene of a single HPV type are multiplelocus assays, even though a single HPV type is detected.

As used herein, “HPV” means human papillomavirus. “HPV” is a generalterm used to refer to any type of HPV, whether currently known orsubsequently described.

As used herein, “fluorophore” refers to a fluorescent reporter moleculewhich, upon excitation with a laser, tungsten, mercury or xenon lamp, ora light emitting diode, releases energy in the form of light with adefined spectrum. Through the process of fluorescence resonance energytransfer (FRET), the light emitted from the fluorophore can excite asecond molecule whose excitation spectrum overlaps the emission spectrumof the fluorophore. The transfer of emission energy of the fluorophoreto another molecule quenches the emission of the fluorophore. The secondmolecule is known as a quencher molecule. The term “fluorophore” is usedinterchangeably herein with the term “fluorescent reporter”.

As used herein “quencher” or “quencher molecule” refers to a moleculethat, when linked to a fluorescent probe comprising a fluorophore, iscapable of accepting the energy emitted by the fluorophore, therebyquenching the emission of the fluorophore. A quencher can befluorescent, which releases the accepted energy as light, ornon-fluorescent, which releases the accepted energy as heat, and can beattached at any location along the length of the probe.

As used herein “dark quencher” refers to a non-fluorescent quencher.

As used herein, “probe” refers to an oligonucleotide that is capable offorming a duplex structure with a sequence in a target nucleic acid, dueto complementarity of at least one sequence of the probe with a sequencein the target region, or region to be detected. The term “probe”includes an oligonucleotide as described above, with or without afluorophore and a quencher molecule attached. The term “fluorescentprobe” refers to a probe comprising a fluorophore and a quenchermolecule.

As used herein, “FAM” refers to the fluorophore 6-carboxy-fluorescein.

Other embodiments of the invention use different oligonucleotidesequences that bind to the E6/E7 gene region of HPV. Theoligonucleotides described herein have a sequence that is capable ofbinding to the target nucleic acid sequence (and its complementarystrand). The oligonucleotides described herein may also be used, eitheralone or in combination, to facilitate detection through amplificationof the HPV E6/E7 gene nucleic acid sequence. In one embodiment, theprobes are designed to perform a Taq-Man® real-time PCR assay on thetarget portion of the gene. Examples of three degenerate probes setsused for Taq-Man® real-time PCR assays, described in terms of theiroligonucleotide sequences, are described below.

Specifically, in the first embodiment where the first degenerateprimer/probe set is for HPV types 39 and 68, the primer/probe set thatprefers HPV type 39 are SEQ ID NOS: 1, 3 and 5. The primer/probe setthat prefers HPV type 68 are SEQ ID NOS. 2, 4 and 6. SEQ ID NOS 1 and 2are both Taq-Man forward primers and are degenerate with respect to eachother. SEQ ID NOS 3 and 4 are both Taq-Man reverse primers and aredegenerate with respect to each other. SEQ ID NOS 5 and 6 are bothTaq-Man probes and are degenerate with respect to each other. Thedegenerate primer/probe sets for HPV types 33 and 58 are SEQ ID NOS.7-16, which include two alternative reverse primer sequences, SEQ IDNOS. 13 and 14 being preferred. The degenerate primer/probe sets for HPVtypes 56 and 66 are SEQ ID NOS. 17-23. The primer/probe sequences thatselect or discriminate for HPV serotype 51 are SEQ ID NOS. 24-29. Theprimer/probe sequences that select or discriminate for HPV Type 59 areSEQ ID NOS. 30-35. All the sequences referenced above are enumerated inTable 1 below. In the table below, “D” is detector, “FP” is forwardprimer, and “RP” is reverse primer.

TABLE 1 SEQ ID NO. Name Description Oligonucleotide Sequence: 5′-3′SEQ ID NO: 1 GR39_68E6 HPV 39 E6  CCACTAGCTGCATGCCAATC FP2 (39) Taq-ManForward Primer SEQ ID NO: 2 GR39_68E6 HPV 68 E6 CCATTAGCTGCATGCCAATCFP2 (68) Taq-Man Forward Primer SEQ ID NO: 3 GR39_68E6 HPV 39 E6CTAATGTAGTTGCATACACCGA RP4 (39) Taq-Man Reverse Primer SEQ ID NO: 4GR39_68E6 HPV 68 E6 CTAATGTTGTTGCATACACCGA RP4 (68) Taq-ManReverse Primer SEQ ID NO: 5 GR39_68D5 HPV 39 E6GAGTAATATCGTAGCTCCCGTATTTT (39) Taq-Man Probe SEQ ID NO: 6 GR39_68D5HPV 68 E6 GAGTAATATCGTAGTTCCCGTATTTT (68) Taq-Man Probe SEQ ID NO: 7GR33_58FP2 HPV 33 E6 TGTGCCAAGCATTGGAGACA (33) Taq-Man Forward PrimerSEQ ID NO: 8 GR33_58FP2 HPV 58 E6 TGTGTCAGGCGTTGGAGACA (58) Taq-ManForward Primer SEQ ID NO: 9 GR33_58RP2 HPV 33 E6 CAAATGGATTTCCCTCTCTATA(33) Taq-Man Reverse Primer SEQ ID NO: 10 GR33_58RP2 HPV 58 E6CAAATGGATTTCCATCTCTATA (58) Taq-Man Reverse Primer SEQ ID NO: 11GR33_58RP3 HPV 33 E6 CCTCTCTATATACAACTGTTAAA (33) Taq-Man Reverse PrimerSEQ ID NO: 12 GR33_58RP3 HPV 58 E6 CCATCTCTATACACTATTCTTAAA (58) Taq-ManReverse Primer SEQ ID NO: 13 GR33_58RP4 HPV 33 E6AAATGGATTTCCCTCTCTATATAC (33) Taq-Man Reverse Primer SEQ ID NO: 14GR33_58RP4 HPV 58 E6 AAATGGATTTCCATCTCTATACAC (58) Taq-ManReverse Primer SEQ ID NO: 15 GR33_58D1 HPV 33 E6TCATATACCTCAGATCGTTGCAAAG (33) Taq-Man Probe SEQ ID NO: 16 GR33_58D1HPV 58 E6 TCATATACCTCAGATCGCTGCAAAG (58) Taq-Man Probe SEQ ID NO: 17MP56_66FP HPV 56 E7 ACCTAATACACGTACCTTGTT (56) Taq-Man Forward PrimerSEQ ID NO: 18 MP56_66FP HPV 66 E7 ACCTAATTCACGTACCTTGTT (66) Taq-ManForward Primer SEQ ID NO: 19 MP56_66RP HPV 56 E7 ACACGCAGGTCCTCTTTGGT(56) Taq-Man Reverse Primer SEQ ID NO: 20 MP56_66RP HPV 66 E7ACACGTAGCTCCTCTTTGGT (66) Taq-Man Reverse Primer SEQ ID NO: 21 MP56_66DHPV 56 E7 TGTAAGTTTGTGGTGCAGTTGGACA (56) Taq-Man Probe SEQ ID NO: 22MP56_66D HPV 56 E7 TGTGAGCTTGTGGTGCAGTTGGACA (66.1) Taq-Man ProbeSEQ ID NO: 23 MP56_66D HPV 66 E7 TGTGAGTTGGTGGTGCAGTTGGACA (66.2)Taq-Man Probe SEQ ID NO: 24 51E6 FP HPV 51 E6 GCAGTATGCAAACAATGTTCACForward Primer SEQ ID NO: 25 51E6 RP HPV 51 E6 TAGTAATTGCCTCTAATGTAGTAReverse Primer SEQ ID NO: 26 51E6 D HPV 51 E6 CCTGCTATAACGTCTATACTCTCTATaq-Man Probe SEQ ID NO: 27 51E7 FP HPV 51 E7 CTCAGAGGAGGAGGATGAAGForward Primer SEQ ID NO: 28 51E7 RP HPV 51 E7 TGAACACCTGCAACACGGAGReverse Primer SEQ ID NO: 29 51E7 D HPV 51 E7 CTACCAGAAAGACGGGCTGGACTaq-Man Probe SEQ ID NO: 30 59E6 FP HPV 59 E6 GGAGAAACATTAGAGGCTGAAForward Primer SEQ ID NO: 31 59E6 RP HPV 59 E6 ATAGAGGTTTTAGGCATCTATAAReverse Primer SEQ ID NO: 32 59E6 D HPV 59 E6 ACCGTTACATGAGCTGCTGATACGTaq-Man Probe SEQ ID NO: 33 59E7 FP HPV 59 E7 GAAGTTGACCTTGTGTGCTACForward Primer SEQ ID NO: 34 59E7 RP HPV 59 E7 ATTAACTCCATCTGGTTCATCTTReverse Primer SEQ ID NO: 35 59E7 D HPV 59 E7 ATTACCTGACTCCGACTCCGAGAATaq-Man Probe SEQ ID NO: 36 Target Region for 91CCATTAGCTGCATGCCAATCATGTATTAAAT Degenerate Primer nucleotideTTTATGCTAAAATACGGGAACTACGATATTA Probe Sets for HPV Target RegionCTCAGAATCGGTGTATGCAACAACATTAG Types 39 and 68 SEQ ID NO: 37Target Region for 138 TGTGCCAAGCATTGGAGACAACTATACACAA Degenerate Primernucleotide CATTGAACTACAGTGCGTGGAATGCAAAAAG Probe Sets for HPVTarget Region ACTTTGCAACGATCTGAGGTATATGATTTTG Types 33 and 58CATTTGCAGATTTAACAGTTGTATATAGAGA GGGAAATCCATTTG SEQ ID NO: 38Target Region for 79 ACCTAATACACGTACCTTGTTGTXAGTGTAA Degenerate Primernucleotide GTTTGTGGTGCAGTTGGACATTCAGAGTACC Probe Sets for HPVTarget Region AAAGAGGACCTGCGTGT Types 56 and 66The locations of the target regions for the primer/probe sets describedin the above table are described in the following Table 1A:

TABLE 1A Location of Target Regions Target Region Genotype GenBankAccession # Coordinates 39 M62849 287-377 68 EU918769 181-271 33 M12732152-288 58 D90400 153-289 56 X74483 747-825 66 EF177190 747-825

While there is sequence homology between the target regions for thedegenerate primer/probe sets, the coordinates of these regions aregenotype dependent.

In a further embodiment, the method includes treating a sample using atleast one degenerate primer/probes set to select for two different butclosely related HPV types and a primer/probe set that discriminates fora third HPV type, where the signal moiety for the degenerate probesemits a signal of the same wavelength and is, preferably, the samesignaling moiety for the two degenerate probes. The signaling moiety ofthe primer/probe set that discriminates for the third HPV type emitssignal at a wavelength that is different, and therefore separatelydetectable, from the wavelength emitted by the signaling moieties forthe degenerate probes. These primer/probe sets are used in a nucleicacid amplification reaction for detecting the presence or absence of theamplified nucleic acid product.

In another embodiment, a kit is provided for the detection of HPV. Thekit includes at least one degenerate primer/probes set that selects fortwo different but closely related HPV types and a primer/probe set thatdiscriminates for a third HPV type, where the signal moiety for thedegenerate probes emits a signal of the same wavelength and is,preferably, the same signaling moiety for the two degenerate probes. Thesignaling moiety of the primer/probe set that discriminates for thethird HPV type emits signal at a wavelength that is different, andtherefore separately detectable, from the wavelength emitted by thesignaling moieties for the degenerate probes. The primer/probe setscapable of amplifying a target sequence that may be used for detectionof that organism. The kit is provided with one or more of theoligonucleotides and buffer reagents for performing amplificationassays.

In yet another aspect of the kit, oligonucleotides and reagents forpurposes of Taq-Man PCR may be provided. In this aspect, threeoligonucleotides are provided. Two of the three are amplificationprimers and the third oligonucleotide is configured as a detector.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the E6 gene target region for the HPV type 68 andschematically, the degeneracy of that target sequence among variousmutations of HPV type 68, HPV type 39 and mutations of HPV type 39 alongwith the degenerate primer/probe set for HPV types 39 and 68;

FIG. 2A-B illustrates the E6 gene target region for the HPV type 33 andschematically, the degeneracy of that target sequence among variousmutations of HPV type 33, HPV type 58 and mutations of HPV type 33 alongwith the degenerate primer/probe set for HPV types 33 and 58; and

FIG. 3 illustrates the E7 gene target for HPV type 66 and schematically,the degeneracy of that target sequence among various mutations of HPVtype 66, HPV type 56 and mutations of HPV type 56 along with thedegenerate primer/probe set for HPV types 56 and 66.

DETAILED DESCRIPTION OF THE INVENTION

The oligonucleotide probes and probes sets described herein arespecifically designed to select for or discriminate between HPV types.Specifically, degenerate primer/probe sets that are somewhat selectivefor one of two closely related HPV types are combined with at least oneother primer/probe set that discriminates for yet a third HPV type thatis different from the closely related HPV types.

The primer/probe sets provide a detectable signal when the specific HPVtype is present in the sample.

In the preferred embodiments, the PCR (e.g. Taq-Man PCR), method ofdetection is used although other methods (e.g. TMA, and LCR) are alsocontemplated. Further, a kit for detecting more HPV types than there arechannels for detection is disclosed.

Referring to FIG. 1, the degenerate primer/probe sets specific for HPVtypes are described in terms of their alignment with the illustratetarget region of the E6 gene. FIG. 1 illustrates the forward primerregion 10, the Taq-Man Detection Region 20 and the reverse primer region30. FIG. 1 also illustrates the degenerate forward primers 11, 12,degenerate reverse primers 31, 32 and degenerate probes 21 and inrelation to their corresponding region on the target sequence 40. Onlythe variations in the target sequence 40 with respect to the forwardprimer region, reverse primer region and detector regions areillustrated in the boxes 60, 70 and 80. Sequence degeneracy betweenthese regions are noted and from that it can be observed that theseregions are less desirable because of increased sequence variation inthese regions.

For example, in box 60 note that there are single nucleotidepolymorphisms (T, C) between the portion of the target sequence 40delimited by box 60 and the same E6 portion of an HPV type 39 target.Boxes 60, 70 and 80 illustrate the nucleotide polymorphisms relative totarget for different strains of HPV types 68 and 39. These differentstrains, and the number and location of the nucleotide polymorphisms(relative to target) are reported in Table 2.

TABLE 2 Polymorphisms Illustrated in FIG. 1 Relative to Target Number ofPolymorphisms Type/Strain (location from 5′) Type 6868_A7_High_45240-DQ80079. seq 3 [T(@6); G(@66); T(@84)]68_A7_High_45240-EU918769.seq None BD115-68 900bp.seq 3 [C(@21); A(@46);C(@81)] BD-637-68 900bp.seq 1 [G(@31)] T276-68 900bp.seq None T1177-68900bp.seq 3 [T or C (@6); C(@81); T or T(@84) T1610-68 900bp 071309.seq2 [G(@53); C(@81)] Type 39 23, A7, High, 10568, 6 [C(@4); A(@27); G(@51) M62849.seq G(@66); C(@69); T(@84)] S492-39 900bp.seq 8 [T(@3);C(@4); A(@27); G (@51) G(@66); C(@69); G(@80); T(@84)] T296-39 900bp.seq7 [C(@4); A(@27); G (@51); C(@57); G(@66); C(@69); T(@84)]

The forward primer 11 for HPV 39 has a single nucleotide polymorphismand is therefore degenerate with respect to the forward primer 12 forHPV 68. Forward primer 11 is SEQ ID NO. 1 and forward primer 12 is SEQID NO. 2. The degeneracy between the two sequences is readily observed:

CCACTAGCTGCATGCCAATC CCATTAGCTGCATGCCAATC

The respective degeneracy is indicated by underlining.

The degeneracy makes forward primer prefer to bind to HPV type 39 andforward primer 12 prefer to bind to HPV type 68. The degenerateprimer/probes sets prefer to bind to the target of one HPV type to theother type but do not discriminate. In terms of the Figures, theprimer/probe sets are described in relation to the target. Boxes 60, 70and 80 contain information about degeneracy among HPV types 39 and 68with respect to the target region 40 delimited by the boxes 60, 70 and80. This is a location reference and not a hybridization reference.Specifically, the reverse primers 31 and 32 (SEQ ID NOs. 3 and 4) havesequences that are the reverse complement of the sequence in theircorresponding location on target 40. Similarly, detector probes 21 and22 (SEQ ID NOS. 5 and 6) are the reverse complement of the sequence intheir corresponding location on target 40. Forward primers 11 and 12(SEQ ID NOS. 1 and 2) are homologous to the sequence in theircorresponding location on target 40.

FIG. 2A-B is similar to FIG. 1, but for degenerate primer/probe setsthat select for HPV types 33 and 58. Boxes 160, 170 and 180 illustratethe nucleotide polymorphisms relative to target for different strains ofHPV types 33 and 58. These different strains, and the number andlocation of the nucleotide polymporphisms (relative to target) arereported in Table 3. The location of the polymorphisms is illustrated inFIG. 2.

TABLE 3 Polymorphisms Illustrated in FIG. 2A-B Relative to TargetType/Strain Number of Polymorphisms Type 33 33_A9_High_10586EF422125.seq 3 [C(@30); A(@62); C(@63)] 33_A9_High_10586 EF422126.seq 3[C(@62); C(@63); T(@52)] 33_A9_High_10586 EF566920.seq 3 [C(@62);C(@63); A(@99)] 33_A9_High_10586 EF566921.seq 2 [C(@62); C(@63)]33_A9_High_10586 EF918766.seq 4 [G(@58); C(@62); C(@63); G(@122)]33_A9_High_10586 GO374550.seq 1 [A(@62)] 33_A9_High_10586 GO374551.seq 2[G(@54); A(@62)] 33_A9_High_10586 GO374552.seq 2 [T(@10); A(@62)]33_A9_High_10586 M12732.seq 2 [A(@62); C(@63)] BD670 grp33 900bp.seq 1[A(@62)] BD783-33 900bp.seq 3 [C(@62); C(@63); C(@92)] BD783-33 clone900bp.seq 2 [C(@62); C(@63)] TI093-33 900bp.seq 2 [G(@54); A(@62)] Type58 HPV_58 D60400.seq 23 [See FIGS. 2A-B for location] T-275-58 900bp.seq22 [See FIGS. 2A-B for location] T-276-58 900bp.seq 24 [See FIGS. 2A-Bfor location] T-817-58 clone 900bp.seq 23 [See FIGS. 2A-B for location]58_A9 High E6 10598 AF478160 23 [See FIGS. 2A-B for location] 58_A9 HighE6 10598 AF478167 22 [See FIGS. 2A-B for location] 58_A9 High E6 10598AF234531 23 [See FIGS. 2A-B for location] 58_A9 High E6 10598 AF47815723 [See FIGS. 2A-B for location] 58_A9 High E6 10598 EU080239 25 [SeeFIGS. 2A-B for location] 58_A9 High E6 10598 FJ407192 24 [See FIGS. 2A-Bfor location] 58_A9 High E6 10598 GO248229 23 [See FIGS. 2A-B forlocation] 58_A9 High E6 10598 GO248253 22 [See FIGS. 2A-B for location]

FIG. 2A-B illustrates the forward primer region 110, the Taq-ManDetection Region 120 and the reverse primer region 130. FIG. 2A-B alsoillustrates the degenerate forward primers 111, 112, degenerate reverseprimers 131, 132 and degenerate probes 121 and 122 in relation to theircorresponding region on the target sequence 140. Only the variations inthe target sequence 140 with respect to the forward primer region,reverse primer region and detector regions are illustrated in the boxes160 (FIG. 2A), 170 and 180 (FIG. 2B).

For example, in box 160 note that there are single nucleotidepolymorphisms (T, G, G) between the portion of the target sequence 140delimited by box 60 and the same E6 portion of an HPV type 33 target.However, the forward primer 112 for HPV 58 has three single nucleotidepolymorphisms and is therefore degenerate with respect to the forwardprimer 111 for HPV 33. Forward primer 111 is SEQ ID NO. 7 and forwardprimer 112 is SEQ ID NO. 8. The degeneracy between the two sequences isreadily observed:

TGTGCCAAGCATTGGAGACA TGTGTCAGGCGTTGGAGACAThe respective degeneracy is indicated by underlining.

The degeneracy makes forward primer 111 prefer to bind to HPV type 33and forward primer 112 prefer to bind to HPV type 58. The degenerateprimer/probes sets prefer to bind to the target of one HPV type to theother type but do not discriminate. In terms of the Figures, theprimer/probe sets are described in relation to the target. Boxes 160,170 and 180 contain information about degeneracy among HPV types 33 and58 with respect to the target region 140 delimited by the boxes 160, 170and 180. This is a location reference and not a hybridization reference.Specifically, the reverse primers 131 and 132 (e.g. SEQ ID NOs. 13 and14) have sequences that are the reverse complement of the sequence intheir corresponding location on target 140. Similarly, reverse probes121 and 122 (SEQ ID NOS. 15 and 16) are the reverse complement of thesequence in their corresponding location on target 140. Forward primers111 and 112 (SEQ ID NOS. 7 and 8) are homologous to the sequence intheir corresponding location on target 140.

FIG. 3 is similar to FIGS. 1 and 2, but for degenerate primer/probe setsthat select for HPV types 56 and 66. FIG. 3 illustrates the forwardprimer region 210, the Taq-Man Detection Region 220 and the reverseprimer region 230. Boxes 260, 270 and 280 illustrate the nucleotidepolymorphisms relative to target for different strains of HPV types 66and 56. These different strains, and the number and location of thenucleotide polymporphisms (relative to target) are reported in Table 2.

TABLE 4 Polymorphisms Illustrated in FIG. 3 Relative to Target Number ofPolymorphisms Type/Strain (location from 5′) Type 6666_A6_High_37119-EF177191 6 [T(@8); A(@24); G(@30) C(@33); G(@71);A(@74)] 66_A6_High_37119-EF177188 7 [T(@8); T(@9); A(@24); G(@30)C(@33); G(@71); A(@74)] 66_A6_High_37119-EF177186 6 [T(@8); A(@24);G(@30) G(@35); G(@71); A(@74)] Type 56 56_A6_High_37119-EF177176 2[G(@24); C(@56)] 56_A6_High_37119-EF177178 3 [C(@20); C(@23); C(@24)]56_A6_High_37119-EF177179 1 [C(@24)] 56_A6_High_37119-EF177180 1[G(@24)] BD616grp56 E7 3[G(@24); T(@54); C(@56)] T1631-56 E7 1 [A(@24)]

FIG. 3 also illustrates the degenerate forward primers 211, 212,degenerate reverse primers 231, 232 and degenerate probes 221 and 222 inrelation to their corresponding region on the target sequence 240. Onlythe variations in the target sequence 240 with respect to the forwardprimer region, reverse primer region and detector regions areillustrated in the boxes 260, 270 and 280.

For example, in box 260 note that there are two single nucleotidepolymorphisms (T, C) between the portion of the target sequence 240delimited by box 260 between and HPV target and an HPV 56 target.However, the forward primer 2112 for HPV 56 has a single nucleotidepolymorphism and is therefore degenerate with respect to the forwardprimer 211 for HPV 66. Forward primer 211 is SEQ ID NO. 17 and forwardprimer 212 is SEQ ID NO. 18. The degeneracy between the two sequences isreadily observed:

ACCTAATACACGTACCTTGTT ACCTAATTCACGTACCTTGTTThe respective degeneracy is indicated by underlining.

The degeneracy makes forward primer 211 prefer to bind to HPV type 56and forward primer 212 prefer to bind to HPV type 66. The degenerateprimer/probes sets prefer to bind to the target of one HPV type to theother type but do not discriminate. In terms of the Figures, theprimer/probe sets are described in relation to the target. Boxes 260,270 and 280 contain information about degeneracy among HPV types 56 and66 with respect to the target region 240 delimited by the boxes 260, 270and 280. This is a location reference and not a hybridization reference.Specifically, the reverse primers 231 and 232 (SEQ ID NOs. 19 and 20)have sequences that are the reverse complement of the sequence in theircorresponding location on target 240. Similarly, probes 221 and 222 (SEQID NOS. 22 and 23) are the reverse complement of the sequence in theircorresponding location on target 240. Forward primers 211 and 212 (SEQID NOS. 17 and 18) are homologous to the sequence in their correspondinglocation on target 140. While not specifically discussed, the degeneracybetween the reverse primers and probes in the degenerate primer probesets is readily observed.

In addition to the examples of degenerate primer/probe sets describedherein, the skilled person, based upon the description herein and theaccompanying figures, would be able to identify other target regions ofclosely related serotypes for which degenerate primer/probe sets couldbe designed. As is readily observed by the Figures, desirable targetregions will have few polymorphisms between the individual serotypes.Those polymorphisms that are present can be addressed in the design ofthe degenerate primer/probe set consistent with the manner describedherein.

As described below, primers and probes can bind to target sequences eventhough they are less than 100% complementary with those regions. Therequisite degree of complementarity depends on a variety of factorsincluding the stringency of the binding conditions. Depending upon thestringency conditions employed, the primers and probes may be modifiedto include different bases in their sequence and still be sufficientlycomplementary to bind to the target region of the nucleic acid.Sufficiently complementary, as used herein include complementarity of70% or more. In preferred embodiments, the complementarity of theprimers/probes to their target sequence is at least 80% over the lengthof the binding portion of the primers/probes. More preferably, thecomplementarity of the primers and probes to their target sequences is90% or more.

Said another way, the present invention contemplates primers and probesthat have at least 70% homology with the primers and probes specificallyidentified herein by SEQ ID. In preferred embodiments, primers/probesthat have at least 80% homology with the primers and probes specificallyidentified by SEQ ID herein are contemplated. More preferably, primersand probes that have at least 90% homology with the primers and probesspecifically identified by SEQ ID herein are contemplated.

While the oligonucleotides described herein must be sufficientlycomplementary to bind their respective portions of the HPV target forwhich they discriminate, it is recognized at some point the sequence ofthe oligonucleotide becomes less complementary to its target and maybind other nucleic acid sequences. Therefore, it is desirable that theoligonucleotide probes remain sufficiently complementary with itsrespective portion of the target gene, and not lose selectivity for itsrespective target binding site.

The target binding sequence within the oligonucleotide amplificationprimer may generally be located at its 3′ end. The target bindingsequence may be about 10-25 nucleotides in length and may havehybridization specificity to the amplification primer. Thus, it isunderstood that one skilled in the art may change the target bindingsequence to effectively change hybridization specificity of theamplification primer and direct hybridization to an alternativesequence.

It is understood to one skilled in the art that the oligonucleotides asused in amplification assays may be modified to some extent without lossof utility or specificity towards a target sequence. For example, as isknown in the art, hybridization of complementary and partiallycomplementary nucleic acid sequences may be obtained by adjustment ofthe hybridization conditions to increase or decrease stringency (i.e.,adjustment of hybridization temperature or salt content of the buffer).Such minor modifications of the disclosed sequences and any necessaryadjustments of hybridization conditions to maintain target-specificityrequire only routine experimentation and are within the ordinary skillin the art.

As a general guide in designing oligonucleotides useful as primers,T_(m) decreases approximately 1° C.-1.5° C. with every 1% decrease insequence homology. Temperature ranges may vary between about 60° C. and70° C., but the primers may be designed to be optimal at 60° C.±4° C.and the probes may be designed to be optimal at 70° C.±4° C. A furtherconsideration when designing amplification primers may be the guanineand cytosine content. Generally, the GC content for a primer may beabout 60-70%, but may also be less and can be adjusted appropriately byone skilled in the art. Annealing complementary and partiallycomplementary nucleic acid sequences may be obtained by modifyingannealing conditions to increase or decrease stringency (i.e., adjustingannealing temperature or salt content of the buffer). Modifications suchas those to the disclosed sequences and any necessary adjustments ofannealing conditions to maintain gene specificity require only routineexperimentation and are within the ordinary skill in the art.

Amplification reactions employing the primers described herein mayincorporate thymine as taught by Walker, et al., supra, or they maywholly or partially substitute 2′-deoxyuridine 5′-triphosphate for TTPin the reaction to reduce cross-contamination of subsequentamplification reactions, e.g., as taught in EP 0 624 643. dU (uridine)is incorporated into amplification products and can be excised bytreatment with uracil DNA glycosylase (UDG). These abasic sites renderthe amplification product not amplifiable in subsequent amplificationreactions. UDG may be inactivated by uracil DNA glycosylase inhibitor(Ugi) prior to performing the subsequent amplification to preventexcision of dU in newly-formed amplification products.

PCR DNA polymerase contemplated for use in the present invention has5′-3′ exonuclease activity (e.g., Sequencing Grade Taq from Promega orDeep Vent® (exo-) DNA from New England BioLabs) are used. The probehybridizes to the target downstream from the PCR amplification primers.The probe is displaced as the downstream endonuclease synthesis proceedsfrom the primers between which the probe is disposed. As thermocyclingis a feature of amplification by PCR, the restriction endonuclease ispreferably added at low temperature after the final cycle of primerannealing and extension for end-point detection of amplification.However, a thermophilic restriction endonuclease which remains activethrough the high temperature phases of the PCR reaction could be presentduring amplification to provide a real-time assay. Linearization of thesecondary structure and separation of the dye pair reduces fluorescencequenching, with a change in a fluorescence parameter such as intensityserving as an indication of target amplification.

The change in fluorescence resulting from unfolding or linearizing ofthe detector oligonucleotides may be detected at a selected endpoint inthe reaction. However, because linearized secondary structures areproduced concurrently with hybridization or primer extension, the changein fluorescence may also be monitored as the reaction is occurring,i.e., in “real-time”. This homogeneous, real-time assay format may beused to provide semi quantitative or quantitative information about theinitial amount of target present. When more initial copies of the targetsequence are present, donor fluorescence more rapidly reaches a selectedthreshold value (i.e., shorter time to positivity). The decrease inacceptor fluorescence similarly exhibits a shorter time to positivity,detected as the time required for reaching a selected minimum value. Inaddition, the rate of change in fluorescence parameters during thecourse of the reaction is more rapid in samples containing higherinitial amounts of target than in samples containing lower initialamounts of target (i.e., increased slope of the fluorescence curve).These or other measurements as is known in the art may be made as anindication of the presence of target or as an indication of targetamplification. The initial amount of target is typically determined bycomparison of the experimental results to results for known amounts oftarget.

Assays for the presence of a selected target sequence according to themethods of the invention may be performed in solution or on a solidphase. Real-time or endpoint homogeneous assays in which the detectoroligonucleotide functions as a primer are typically performed insolution. Hybridization assays using the detector oligonucleotides ofthe invention may also be performed in solution (e.g., as homogeneousreal-time assays) but are also particularly well-suited to solid phaseassays for real-time or endpoint detection of target. In a solid phaseassay, detector oligonucleotides may be immobilized on the solid phase(e.g., beads, membranes or the reaction vessel) via internal or terminallabels using methods known in the art. For example, a biotin-labeleddetector oligonucleotide may be immobilized on an avidin-modified solidphase where it will produce a change in fluorescence when exposed to thetarget under appropriate hybridization conditions. Capture of the targetin this manner facilitates separation of the target from the sample andallows removal of substances in the sample which may interfere withdetection of the signal or other aspects of the assay.

For commercial convenience, oligonucleotides useful for specificdetection and identification of HPV nucleic acids may be packaged in theform of a kit. Typically, such a kit contains at least oneoligonucleotide described herein. Reagents for performing a nucleic acidamplification reaction may also be included with the HPV-specificoligonucleotides. For example, buffers, other oligonucleotides,nucleotide triphosphates, enzymes, etc. may be included. The componentsof the kit may be packaged together in a common container. Optionallyinstructions may be included that illustrate one described embodimentfor performing a specific embodiment of the inventive methods. Otheroptional components may also be included in the kit, e.g., anoligonucleotide tagged with a label suitable for use as an assay probe,and/or reagents or means for detecting the label.

Furthermore, the kit may include oligonucleotides and reagents in driedor liquid format. The components of the kit may be more stable andeasily manipulated when in dried format. The dried components of the kitmay be added or pre-treated to a solid phase such as microtiter plate,microarray, or other appropriate receptacle, where the sample and bufferneed only be added. This format facilitates assaying multiple samplessimultaneously and is useful in high-throughput methods. The BDProbeTec™ and Viper™ XTR instruments may be used.

The following Examples illustrate specific embodiments of the inventiondescribed herein. As would be apparent to skilled artisans, variouschanges and modifications are possible, and are contemplated within thescope of the invention described.

A Taq-Man PCR System for Detecting HPV is further described below usingthe primer/probes sets in Table 1 as an Example. The Primer Sets ofPrimers/Probes described in Table 1 above were designed to performTaq-Man PCR on for HPV types 39 and 68 and 51. Specifically, in thefirst embodiment where the first degenerate primer/probe set is for HPVtypes 39 and 68, the primer/probe set that prefers HPV type 39 are SEQID NOS: 1, 3 and 5. The primer/probe set that prefers HPV type 68 areSEQ ID NOS. 2, 4 and 6.

Taq-Man real-time PCR is a type of quantitative PCR. Taq-Man uses afluorogenic probe which is a single stranded oligonucleotide of 20-26nucleotides and is designed to bind only the DNA sequence between thetwo PCR primers. In Taq-Man, reporter dyes and quencher dyes areattached to the probe. The probe is annealed to the DNA by alternatingthe temperature to denature and re-anneal the DNA. The Taq polymeraseadds nucleotides to the target DNA and this removes the Taq-Man probefrom the template DNA. When the reporter dye is separated from thequencher dye, the reporter dye emits energy which is detectable. Theenergy is quantified by a computer, which provides a signal indicatingthat the target was detected. Only the specific PCR product can generatethe fluorescent signal in Taq-Man PCR.

In the example herein, thermal cycling is contemplated. After an initialdenature step at 95° C. for 15 minutes, the PCR mixture of primer/probesets and sample for the detection of the presence or absence of targetis subjected to the thermal cycle of 55° C. for 1 minute followed by 95°C. for 30 seconds for forty cycles.

To practice Taq-Man PCR, two PCR primers with a preferred product sizeof 50-150 base pairs and a probe with a fluorescent reporter orfluorophore (e.g. 6-carboxyfluorescein (FAM) and tetrachlorofluorescin(TET)) and a quencher such as tetramethylrhodamine (TAMRA) or a darkquencher such as previously described is covalently attached to its 5′and 3′ ends are used. Suitable fluorescent reporters and fluorophoresare well known and not described in detail herein.

TABLE 5 Examples of Taq-Man PCR Probes Sets for Taq-ManAssay of HPV Types 39, 68 and 51 ORF Location (bp) Genbank Accession inSEQ ID NO: Probe description Oligonucleotide 5′ Sequence 3′ ()SEQ ID NO: 1 HPV 39 E6 CCACTAGCTGCATGCCAATC 287-306 Taq-Man (M62849)Forward Primer SEQ ID NO: 2 HPV 68 E6 CCATTAGCTGCATGCCAATC 181-200Taq-Man (EU918769) Forward Primer SEQ ID NO: 3 HPV 39 E6CTAATGTAGTTGCATACACCGA 356-377 Taq-Man (M62849) Reverse PrimerSEQ ID NO: 4 HPV 68 E6 CTAATGTTGTTGCATACACCGA 250-271 Taq-Man (EU918769)Reverse Primer SEQ ID NO: 5 HPV 39 E6 GAGTAATATCGTAGCTCCCGTATTTT 326-351Taq-Man (M62849) Probe SEQ ID NO: 6 HPV 68 E6 GAGTAATATCGTAGTTCCCGTATTTT220-245 Taq-Man (EU918769) Probe SEQ ID NO: 24 HPV 51 E6GCAGTATGCAAACAATGTTCAC 277-298 Forward Primer (M62866) SEQ ID NO: 25HPV 51 E6 TAGTAATTGCCTCTAATGTAGTA 351-373 Reverse Primer (M62877)SEQ ID NO: 26 HPV 51 E6 CCTGCTATAACGTCTATACTCTCTA 315-339 Taq-Man(M62877) Probe SEQ ID NO: 27 HPV 51 E7 CTCAGAGGAGGAGGATGAAG 652-671Forward Primer (M62877) SEQ ID NO: 28 HPV 51 E7 TGAACACCTGCAACACGGAG738-757 Reverse Primer (M62877) SEQ ID NO: 29 HPV 51 E7CTACCAGAAAGACGGGCTGGAC 692-713 Taq-Man (M62877) Probe

The probes are designed to anneal to the ORF location in the HPV E6/E7gene that is noted in the Table. In this regard, the ORF locations forthe primer probe/set for HPV 59 for both the E6 and E7 genes are listedin the following table.

TABLE 6 ORF Locations of E6/E7 for Primer/Probe Set for HPV Type 59ORF Location (bp) Genbank Accession in SEQ ID NO: Probe descriptionOligonucleotide 5′ Sequence 3′ () SEQ ID NO: 30 HPV 59 E6GGAGAAACATTAGAGGCTGAA 313-333 Forward Primer (X77858) SEQ ID NO: 31HPV 59 E6 ATAGAGGTTTTAGGCATCTATAA 369-391 Reverse Primer (X77858)SEQ ID NO: 32 HPV 59 E7 ACCGTTACATGAGCTGCTGATACG 342-365 Taq-Man(X77858) Probe SEQ ID NO: 33 HPV 59 E7 GAAGTTGACCTTGTGTGCTAC 605-625Forward Primer (X77858) SEQ ID NO: 34 HPV 59 E7 ATTAACTCCATCTGGTTCATCTT660-682 Reverse Primer (X77858) SEQ ID NO: 35 HPV 59 E7ATTAACTCCATCTGGTTCATCTT 631-654 Taq-Man (X77858) Probe

In addition to the primers and probes, Taq-Man PCR requires reagentsthat are used for regular PCR (e.g. polymerase, free nucleotides) aswell as a real-time PCR machine for analyzing the data. The reagents andequipment are well known to those skilled in the art and are notdiscussed in detail herein.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of theinvention described herein. It is therefore to be understood thatnumerous modifications may be made to the illustrative embodiments andthat other arrangements may be devised without departing from the spiritand scope of the invention described herein as defined by the appendedclaims.

The invention claimed is:
 1. A multiplex assay for detecting thepresence or absence of at least one serotype of human papillomavirus(HPV) in a biological sample comprising: providing a biological sample;contacting the biological sample with at least three oligonucleotideprimer/probe sets, each of which comprises at least one oligonucleotideprobe that is detectably labeled and has a nucleotide sequence length ofabout 10 to about 50 and at least two oligonucleotide primers each ofwhich has a nucleotide sequence length of about 10 to about 150 underconditions such that the probes and primers anneal to a target sequenceand wherein each primer/probe set is at least preferential for aspecific serotype of an organism wherein the first and secondprimer/probe sets are degenerate with respect to each other and whereinthe degenerate probes each have a signal moiety that emits signal thatis detectable on the same channel and wherein the third primer/probe setis not degenerate with respect to the other two primer/probe sets anddiscriminates for a third serotype that is not the serotypes to whichthe degenerate primer/probe sets preferentially anneal and wherein thethird primer/probe set has a signal moiety that emits signal at awavelength that is the same or different from the wavelength emitted bythe signal moiety of the degenerate probes; wherein the target for thedegenerate primer/probe set is SEQ ID NO: 36; amplifying, if present,the target sequence; and monitoring for detection the label as anindication of the hybridization of the probe set to the target sequencethereby indicating the presence or absence of at least one of theserotypes of HPV.
 2. The assay of claim 1, further comprising contactingthe biological sample with at least one additional degenerateprimer/probe set wherein the target for the additional degenerateprimer/probe set is selected from the group consisting of SEQ ID NO: 37and
 38. 3. The assay of claim 1 wherein the third primer/probe set isconfigured to discriminate for HPV serotype 51 and is not degeneratewith respect to the first or second primer/probe set, wherein the thirdprimer/probe set is selected from at least one of the sequences selectedfrom the group consisting of SEQ ID NO. 24-29 and sequences that are atleast 70% homologous to SEQ ID NO. 24-29; the first primer/probe setselects for HPV serotype 39 and does not discriminate for HPV 39serotype with respect to HPV serotype 68, wherein the first primer/probeset is selected from at least one of the sequences selected from thegroup consisting of SEQ ID NO. 1, 3 and 5 and sequences that are atleast 70% homologous to SEQ ID NO. 1, 3 and 5; and the secondprimer/probe set selects for HPV serotype 68 and does not discriminatefor HPV 68 serotype with respect to HPV serotype 39, wherein the secondprimer/probe set is selected from at least one of the sequences selectedfrom the group consisting of SEQ ID NO. 2, 4 and 6 and sequences thatare at least 70% homologous to SEQ ID NO. 2, 4 and
 6. 4. The assay ofclaim 2 wherein the degenerate primer/probe set target is SEQ ID NO. 37and wherein the third primer/probe set is configured to discriminate forHPV serotype 59 and is not degenerate with respect to the first orsecond primer/probe set, wherein the third primer/probe set is selectedfrom at least one of the sequences selected from the group consisting ofSEQ ID NO. 30-35 and sequences that are at least 70% homologous to SEQID NO. 30-35; the first primer/probe set selects for HPV serotype 33 anddoes not discriminate for HPV 33 serotype with respect to HPV serotype58, wherein the first primer/probe set is selected from at least of thesequences selected from the group consisting of SEQ ID NO. 7, 9, 11, 13,and 15 and sequences that are at least 70% homologous to SEQ ID NO. 7,9, 11, 13, and 15; and the second primer/probe set selects for HPVserotype 58 and does not discriminate for HPV 58 serotype with respectto HPV serotype 33, wherein the second primer/probe set is selected fromat least one of the sequences selected from the group consisting of SEQID NO. 8, 10, 12, 14, and 16 and sequences that are at least 70%homologous to SEQ ID NO. 8, 10, 12, 14, and
 16. 5. The assay of claim 2wherein the degenerate primer/probe set target is SEQ ID NO. 38 andwherein the third primer/probe set is configured to discriminate for HPVserotype 59 and is not degenerate with respect to the first or secondprimer/probe set, wherein the third primer/probe set is selected from atleast one of the sequences selected from the group consisting of SEQ IDNO. 30-35 and sequences that are at least 70% homologous to SEQ ID NO.30-35; the first primer/probe set selects for HPV serotype 56 and doesnot discriminate for HPV 66 serotype with respect to HPV serotype 56,wherein the first primer/probe set is selected from at least of thesequences selected from the group consisting of SEQ ID NO. 17, 19 and 21and sequences that are at least 70% homologous to SEQ ID NO. 17, 19 and21; and the second primer/probe set selects for HPV serotype 66 and doesnot discriminate for HPV 66 serotype with respect to HPV serotype 56,wherein the second primer/probe set is selected from at least of thesequences selected from the group consisting of SEQ ID NO. 18, 20, 22,and 23 and sequences that are at least 70% homologous to SEQ ID NO. 18,20, 22, and
 23. 6. The assay of claim 1 wherein the third primer/probeset is configured to discriminate for HPV serotype 51 and is notdegenerate with respect to the first or second primer/probe set, whereinthe third primer/probe set is selected from at least one of thesequences selected from the group consisting of SEQ ID NO. 24-29 andsequences that are at least 80% homologous to SEQ ID NO. 24-29; thefirst primer/probe set selects for HPV serotype 39 and does notdiscriminate for HPV 39 serotype with respect to HPV serotype 68,wherein the first primer/probe set is selected from at least one of thesequences selected from the group consisting of SEQ ID NO. 1, 3 and 5and sequences that are at least 80% homologous to SEQ ID NO. 1, 3 and 5;and the second primer/probe set selects for HPV serotype 68 and does notdiscriminate for HPV 68 serotype with respect to HPV serotype 39,wherein the second primer/probe set is selected from at least one of thesequences selected from the group consisting of SEQ ID NO. 2, 4 and 6and sequences that are at least 80% homologous to SEQ ID NO. 2, 4 and 6.7. The assay of claim 2 wherein the degenerate primer/probe set targetis SEQ ID NO. 37 and wherein the third primer/probe set is configured todiscriminate for HPV serotype 59 and is not degenerate with respect tothe first or second primer/probe set, wherein the first primer/probe setis selected from at least one of the sequences selected from the groupconsisting of SEQ ID NO. 30-35 and sequences that are at least 80%homologous to SEQ ID NO. 30-35; the first primer/probe set selects forHPV serotype 33 and does not discriminate for HPV 33 serotype withrespect to HPV serotype 58, wherein the first primer/probe set isselected from at least of the sequences selected from the groupconsisting of SEQ ID NO. 7, 9, 11, 13, and 15 and sequences that are atleast 80% homologous to SEQ ID NO. 7, 9, 11, 13, and 15; and the secondprimer/probe set selects for HPV serotype 58 and does not discriminatefor HPV 58 serotype with respect to HPV serotype 33, wherein the secondprimer/probe set is selected from at least one of the sequences selectedfrom the group consisting of SEQ ID NO. 8, 10, 12, 14, and 16 andsequences that are at least 80% homologous to SEQ ID NO. 8, 10, 12, 14,and
 16. 8. The assay of claim 2 wherein the degenerate primer/probe settarget is SEQ ID NO. 38 and wherein the third primer/probe set isconfigured to discriminate for HPV serotype 59 and is not degeneratewith respect to the first or second primer/probe set, wherein the thirdprimer/probe set is selected from at least one of the sequences selectedfrom the group consisting of SEQ ID NO. 30-35 and sequences that are atleast 80% homologous to SEQ ID NO. 30-35; the first primer/probe setselects for HPV serotype 56 and does not discriminate for HPV 66serotype with respect to HPV serotype 56, wherein the first primer/probeset is selected from at least of the sequences selected from the groupconsisting of SEQ ID NO. 17, 19 and 21 and sequences that are at least80% homologous to SEQ ID NO. 17, 19 and 21; and the second primer/probeset selects for HPV serotype 66 and does not discriminate for HPV 66serotype with respect to HPV serotype 56, wherein the secondprimer/probe set is selected from at least of the sequences selectedfrom the group consisting of SEQ ID NO. 18, 20, 22, and 23 and sequencesthat are at least 80% homologous to SEQ ID NO. 18, 20, 22, and
 23. 9.The assay of claim 1 wherein the third primer/probe set is configured todiscriminate for HPV serotype 51 and is not degenerate with respect tothe first or second primer/probe set, wherein the third primer/probe setis selected from at least one of the sequences selected from the groupconsisting of SEQ ID NO. 24-29 and sequences that are at least 90%homologous to SEQ ID NO. 24-29; the first primer/probe set selects forHPV serotype 39 and does not discriminate for HPV 39 serotype withrespect to HPV serotype 68, wherein the first primer/probe set isselected from at least one of the sequences selected from the groupconsisting of SEQ ID NO. 1, 3 and 5 and sequences that are at least 90%homologous to SEQ ID NO. 1, 3 and 5; and the second primer/probe setselects for HPV serotype 68 and does not discriminate for HPV 68serotype with respect to HPV serotype 39, wherein the secondprimer/probe set is selected from at least one of the sequences selectedfrom the group consisting of SEQ ID NO. 2, 4 and 6 and sequences thatare at least 90% homologous to SEQ ID NO. 2, 4 and
 6. 10. The assay ofclaim 2 wherein the degenerate primer/probe set target is SEQ ID NO. 37and wherein the third primer/probe set is configured to discriminate forHPV serotype 59 and is not degenerate with respect to the first orsecond primer/probe set, wherein the primer/probe set is selected fromat least one of the sequences selected from the group consisting of SEQID NO. 30-35 and sequences that are at least 90% homologous to SEQ IDNO. 30-35; the first primer/probe set selects for HPV serotype 33 anddoes not discriminate for HPV 33 serotype with respect to HPV serotype58, wherein the first primer/probe set is selected from at least of thesequences selected from the group consisting of SEQ ID NO. 7, 9, 11, 13,and 15 and sequences that are at least 90% homologous to SEQ ID NO. 7,9, 11, 13, and 15; and the second primer/probe set selects for HPVserotype 58 and does not discriminate for HPV 58 serotype with respectto HPV serotype 33, wherein the second primer/probe set is selected fromat least one of the sequences selected from the group consisting of SEQID NO. 8, 10, 12, 14, and 16 and sequences that are at least 90%homologous to SEQ ID NO. 8, 10, 12, 14, and
 16. 11. The assay of claim 2wherein the degenerate primer/probe set target is SEQ ID NO. 38 andwherein the third primer/probe set is configured to discriminate for HPVserotype 59 and is not degenerate with respect to the first or secondprimer/probe set, wherein the primer/probe set is selected from at leastone of the sequences selected from the group consisting of SEQ ID NO.30-35 and sequences that are at least 90% homologous to SEQ ID NO.30-35; the first primer/probe set selects for HPV serotype 56 and doesnot discriminate for HPV 66 serotype with respect to HPV serotype 56,wherein the first primer/probe set is selected from at least of thesequences selected from the group consisting of SEQ ID NO. 17, 19 and 21and sequences that are at least 90% homologous to SEQ ID NO. 17, 19 and21; and the second primer/probe set selects for HPV serotype 66 and doesnot discriminate for HPV 66 serotype with respect to HPV serotype 56,wherein the second primer/probe set is selected from at least of thesequences selected from the group consisting of SEQ ID NO. 18, 20, 22,and 23 and sequences that are at least 90% homologous to SEQ ID NO. 18,20, 22, and 23.